p21 (WAF1/CIP1) is a downstream effector of p53 and mediates growth arrest by inhibiting the action of G 1 cyclin-dependent kinases. However, it has been reported that the p21 expression was triggered by multiple differentiation-inducing agents by a p53-independent pathway. These agents induced expression of p21 by binding to specific DNA elements and modulating transcriptional initiation. We demonstrated that the gene encoding p21 was not only a vitamin D 3 target gene but also a vitamin K 2 target gene in the cells and that their differentiation was well related to the transcriptional activation of the p21 gene. Transient overexpression of p21, using adenovirus-driven p21 expression plasmid, in MG-63 cells in the absence of vitamins D 3 and K 2 resulted in their differentiation. The transcriptional activation of p21 by vitamin D 3 or vitamin K 2 in p53-deficient osteosarcoma cells demonstrated the p53-independent role of p21 in human osseous differentiation. HUMPATHOL32:410-416. Copyright (C) 2001 by W.B. Saunders Company